Plasma phospholipid transfer protein (PLTP) plays a key role in lipid and lipoprotein metabolism. Known functions of PLTP include transfer of phospholipids (PL) and cholesterol among lipoproteins and between lipoproteins and cells, modulation of HDL level, size, and composition, and enhancement of cellular cholesterol efflux. Current data indicate that most PLTP in human plasma associates with distinct HDL particles and lacks the ability to transfer PL from liposomes to HDL in vitro. The physiological significance of this inactive form of PLTP is unknown. We recently found that PLTP interacts with cell surface ABCA1 and promotes transport of cell cholesterol and PL to HDL particles. The structural domains of PLTP and ABCA1 involved in this process remain to be determined. The major goals of this proposal are to establish the structural basis and the metabolic and pathologic relevance of inactive PLTP, and to define the interaction of PLTP with cellular ABCA1 and its role in reverse cholesterol transport. Our overall hypothesis is that specific structural features of PLTP are critical for lipid transport and for interacting with ABCA1. To test this hypothesis, we will determine the biochemical characteristics of lipoprotein particles containing active and inactive PLTP, delineate their potential molecular differences, examine the possibility of their interconversion, and study their roles in modulating plasma lipoproteins and in cellular lipid homeostasis. We will also characterize the mechanisms of PLTP-mediated lipid removal by the ABCA1 pathway, and define molecular properties of PLTP and ABCA1 required for their interaction and lipid efflux using PLTP domain- specific peptides, and PLTP and ABCA1 mutants. Furthermore, we will investigate the pathological relevance of the balance between active and inactive PLTP in the pathogenesis of FCHL and the quantitative trait loci for PLTP activity and active and inactive PLTP mass in families with FCHL in collaboration with Project 1. The proposed studies should provide novel insights into the mechanisms of action and the role of PLTP in lipoprotein metabolism and pathophysiology. A better understanding of the basic function and mechanisms of action of PLTP could lead to more targeted treatment and prevention of lipoprotein disorders that contribute to the development of cardiovascular disease.